The overall goal of this researches to design strategies for cartilage repair. Specifically, we will create novel materials derived from glucosamine and determine the mechanism of glucosamine action on chondrocytes and bone marrow derived stem cells. Glucosamine has had a long history in clinical treatment of cartilage degeneration. Unfortunately there has been conflicting validation of efficacy in vitro and little understanding of glucosamine's mechanism of action. Some researchers have hypothesized that glucosamine functions to directly increase cartilage polysaccharide synthesis. The expertise of a glycobiologist is critical to finally understanding glucosamine activity on cartilage. Our preliminary data and coPI expertise demonstrates that glucosamine functions by O-Glc-NAc protein modification which in turn regulates numerous cell activities to increase matrix production. We have also demonstrated increased cartilage extracellular matrix deposition in hydrogels when engineering cartilage from chondrocytes and mesenchymal stem cells (MSCs) in the presence of glucosamine. Results of this study will allow us to definitively provide the mechanism of glucosamine action on cartilage growth and design appropriate repair strategies. This proposal is both hypothesis driven and design driven. We hypothesize that glucosamine enters the metabolic hexosamine pathway in cells that supports O-GlcNAc-mediated cell responses to improve new cartilage formation instead of being directly incorporated into glycosaminoglycan synthesis as previously hypothesized by researchers. In the design portion of the proposal we will investigate chondrocyte and MSC response to glucosamine in hydrogel tissue engineering systems. Furthermore, we will develop novel materials derived from glucosamine so that the molecule can be readily released from a biomaterial scaffold for in vivo delivery. The following specific aims will address these research topics. Specific aim 1. Evaluate glucosamine activity on cartilage tissue engineering 1A: Determine glucosamine influence on cartilage tissue formation by chondrocytes and MSCs. 1B: Identify specific biochemical pathways responsible for cellular responses to glucosamine. Specific aim 2. Synthesize a polymeric glucosamine for incorporation into 3D scaffolds for cartilage engineering. . [unreadable] [unreadable] [unreadable]